Resolving the native conformation of Escherichia coli OmpA.

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain structure. Our recent studies show that Ser163 and Ser167 of the N-terminal domain (1-170) are modified in the cytoplasm by covalent attachment of oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N-terminal domain to be incorporated into planar lipid bilayers as narrow pores (≈ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M-OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I-OmpA). Chemical and Western blot analysis and 1H-NMR showed that segment 264-325 in M-OmpA, but not in I-OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M-OmpA undergo a temperature-induced transition into stable large pores (≈ 450 pS, 1 M KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol(-1)], but this transition does not occur with I-OmpA or with M-OmpA that has been exposed to disulfide bond-reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB-modification, disulfide bond formation and temperature in folding OmpA into its native large-pore configuration.